Human prostate cell lines in cancer treatment

ABSTRACT

Combinations of cell lines are provided for allogeneic immunotherapy agents in the treatment of cancer. Cancer vaccines generally have been limited to the use of cells that contain at least some tumor specific antigens (“TSAs”) and/or tumor associated antigens (“TAAs”) having shared identity with antigens in a targeted tumor. In such cases, tumor cells often are utilized as a starting point on the premise that only tumor cells will contain TSAs or TAAs or relevance, and the tissue origins of the cells are matched to the tumor site in patients. A primary aspect of the invention is the use of immortalised normal, non-malignant cells, in combination with primary and/or metastatic tumor cells, as the basis of an allogeneic cell cancer vaccine. Normal cells do not posses TSAs or relevant concentrations of TAAs and hence it is surprising that normal cells are effective as anti-cancer vaccines when administered in combination with primary and/or metastatic tumor cells. More surprisingly, a three way combination of cells obtained from metastasized cells, non metastasized tumor and cells from a normal cell line provided good therapy. For prostate cancer, for example, a vaccine may be based on one or a combination of different immortalized normal cell lines derived from the prostate according to parameters described herein. The cell lines may be lethally irradiated with, for example, gamma irradiation at 50-300 Gy to ensure that they are replication incompetent prior to use.

This application is a continuation of U.S. application Ser. No.10/897,426, filed Jul. 23, 2004, now allowed, which is a continuation inpart of U.S. application Ser. No. 10/624,889, filed Jul. 23, 2003, theentireties of which are hereby incorporated by reference.

FIELD OF THE INVENTION

The invention relates to the treatment of primary, metastatic, andresidual cancer in mammals, and more particularly to the use ofmaterials such as whole cells and derivatives and portions thereof tostimulate the immune system to attack cancer.

BACKGROUND TO THE INVENTION

Cancerous cells contain numerous mutations that can result inrecognition of the cells by a host's immune system. Appreciation of thisphenomenon has prompted much research into potential immunotherapies toharness the host's immune system for attacking cancer cells. Eliminatingthese cells or reducing them to a level that is not life-threatening hasbeen a major goal, as reviewed in Maraveyas, A. & Dalgleish, A. G. 1997Active immunotherapy for solid tumours in vaccine design in The Role ofCytokine Networks, Ed. Gregoriadis et al., Plenum Press, New York, pages129-145; Morton, D. L. and Ravindranath, M. H. 1996 Current conceptsconcerning melanoma vaccines in Tumor Immunology—Immunotherapy andCancer Vaccines, ed. Dalgleish, A. G. and Browning, M., CambridgeUniversity Press, pages 241-268.

Such work in the cancer immunotherapy field can be classified into fivecategories, non-specific immunotherapy, antibodies and monoclonalantibodies, subunit vaccines, gene therapy, and cell-based vaccines.

Non-Specific Immunotherapy

Efforts to stimulate the immune system non-specifically date back over acentury to the pioneering work of William Coley (Coley, W. B., 1894Treatment of inoperable malignant tumours with toxins of erysipelas andthe Bacillus prodigosus. Trans. Am. Surg. Assoc. 12: 183). Althoughsuccessful in a limited number of cases (e.g. BCG (i.e. bacilleCalmette-Guérin) for the treatment of urinary bladder cancer, IL-2 forthe treatment of melanoma and renal cancer) it is widely acknowledgedthat non-specific immunomodulation is unlikely to prove sufficient totreat the majority of cancers. While non-specific immune-stimulants maylead to a general enhanced state of immune responsiveness, they lack thetargeting capability and also subtlety to deal with tumour lesions whichhave many mechanisms and plasticity to evade, resist and subvertimmune-surveillance.

Antibodies and Monoclonal Antibodies

Passive immunotherapy in the form of antibodies, and particularlymonoclonal antibodies, has been the subject of considerable research anddevelopment as anti-cancer agents. Originally hailed as the magic bulletbecause of their exquisite specificity, monoclonal antibodies havefailed to live up to their expectation in the field of cancerimmunotherapy for a number of reasons, including immune responses to theantibodies themselves and inability of the antibody to access the lesionthrough the blood vessels (thereby abrogating their activity). To date,few products have been registered as pharmaceuticals for human use,notably Rituxan (IDEC/Genentech/Hoffman la Roche) and Herceptin(Genentech/Hoffman la Roche) with over 50 other projects in the researchand development pipeline. Antibodies also may be employed in activeimmunotherapy utilizing anti-idiotype antibodies which appear to mimic(in an immunological sense) cancer antigens. Although elegant inconcept, the utility of antibody-based approaches may ultimately provelimited by the phenomenon of ‘immunological escape,’ where a subset ofcancer cells in a mammalian or human subject mutates and loses theantigen recognized by the particular antibody and thereby can lead tothe outgrowth of a population of cancer cells that are no longertreatable with that antibody.

Subunit Vaccines

Drawing on the experience in vaccines for infectious diseases and otherfields, many researchers have sought to identify antigens that areexclusively or preferentially associated with cancer cells, namelytumour specific antigens (TSA) or tumour associated antigens (TAA), andto use such antigens or fractions thereof as the basis for specificactive immunotherapy.

There are numerous ways to identify proteins or peptides derivedtherefrom which fall into the category of TAA or TSA. For example, it ispossible to utilize differential display techniques whereby RNAexpression is compared between tumour tissue and adjacent normal tissueto identify RNAs which are exclusively or preferentially expressed inthe lesion. Sequencing of the RNA has identified several TAA and TSAwhich are expressed in that specific tissue at that specific time, buttherein lies the potential deficiency of the approach in thatidentification of the TAA or TSA represents only a “snapshot” of thelesion at any given time which may not provide an adequate reflection ofthe antigenic profile in the lesion over time. Similarly a combinationof cytotoxic T lymphocyte (CTL) cloning and expression-cloning of cDNAfrom tumour tissue has lead to identification of many TAA and TSA,particularly in melanoma. The approach suffers from the same inherentweakness as differential display techniques in that identification ofonly one TAA or TSA may not provide an appropriate representation of aclinically relevant antigenic profile.

Over fifty subunit vaccine approaches are in development for treating awide range of cancers, although none has yet received marketingauthorization for use as a human pharmaceutical product. In a similarmanner to that described for antibody-based approaches above, subunitvaccines also may be limited by the phenomenon of immunological escape.

Gene Therapy

Most gene therapy trials in humans concern cancer treatment. Asubstantial proportion of these trial have purported to trigger and/oramplify patients' immune responses. Of particular note in areAllovectin-7 and Leuvectin, developed by Vical Inc for a range of humantumours, and StressGen Inc.'s stress protein gene therapy for melanomaand lung cancer. It is too early to judge whether these and the other‘immuno-gene therapies’ in development by commercial and academic bodiesultimately will prove successful. However the commercial utility ofthese approaches are expected to be more than a decade away.

Cell-Based Vaccines

Tumours have the remarkable ability to counteract the immune system in avariety of ways. These include, downregulating the expression ofpotential target proteins; mutation of potential target proteins;downregulating surface expression of receptors and other proteins;downregulating MHC class I and II expression thereby hindering directpresentation of TAA or TSA peptides; downregulating co-stimulatorymolecules leading to incomplete stimulation of T-cells and thus toanergy; shedding of selective, non representative membrane portions thatact as decoys to the immune system; shedding of selective membraneportions that anergise the immune system; secreting inhibitorymolecules; inducting T-cell death; and other ways. Because of this widediversity of escape mechanisms, their immunological heterogeneity andplasticity, tumours growth has to be matched with suitableimmunotherapeutic strategies that can account for such heterogeneity.The potential advantages are:

-   -   (a) whole cells contain a broad range of antigens, providing an        antigenic profile of sufficient heterogeneity to match that of        the lesions as described above;    -   (b) being multivalent (i.e. containing multiple antigens), the        risk of immunological escape is reduced (the probability of        cancer cells ‘losing’ all of these antigens is remote); and    -   (c) cell-based vaccines include TSAs and TAAs that have yet to        be identified as such; it is possible if not likely that        currently unidentified antigens may be clinically more relevant        than the relatively small number of TSAs/TAAs that are known.

Cell-based vaccines fall into two categories. The first category usesautologous cells. Typically a procedure within this category begins withtaking a biopsy from a patient, cultivating tumour cells from the biopsyin vitro, modifying the cultivated cells through transfection and/orother means, irradiating the modified cells to render themreplication-incompetent, and then injecting the replication-incompetentcells back into the same patient as a vaccine. Although this approachenjoyed considerable attention over the past decade, it has beenincreasingly apparent that this individually-tailored therapy isinherently impractical for several reasons. The procedure is timeconsuming as the lead time for producing clinical doses of vaccine oftenmay exceed the patients' life expectancy. The procedure may be expensiveand, as a ‘bespoke’ product, it is not possible to specify astandardised product (only the procedure, not the product, can bestandardised and hence optimised and quality controlled). Still further,the tumour biopsy used to prepare the autologous vaccine generally willhave unique growth characteristics, interactions and communications withsurrounding tissue. The characteristics of the initial cell sample,which reflect a particular environment at a single time point from atumour may severely limit the use of autologous cells for immunotherapy,wherein a vaccine desirably may be administered over the entirepresentation time of a disease.

The second category of cell-based vaccines utilize allogeneic cells.These vaccines comprise cells that that genetically (and henceimmunologically) are mismatched to patients. Allogeneic cell proceduresbenefit from the same advantages of multivalency as autologous cells. Inaddition, allogeneic cell vaccines can utilize immortalized cell lines,which can be cultivated indefinitely in vitro. Thus, this approachovercomes the lead-time and cost disadvantages of autologousmethodologies.

Numerous publications extol the utility of cell-based cancer vaccines.See, for example, Dranoff, G. et al. WO 93/06867; Gansbacher, P. WO94/18995; Jaffee, E. M. et al. WO 97/24132; Mitchell, M. S. WO 90/03183;and Morton, D. M. et al. WO 91/06866. These studies report proceduralvariations that range from a basic technique of using cancer cells as animmunotherapy antigen, to transfecting the cells to produce GM-CSF,IL-2, interferons or other immunologically-active molecules to the useof ‘suicide’ genes. Various research groups have reported the use ofallogeneic cell lines for use against melanoma, that are HLA-matched orpartially-matched to a patients' haplotype and allogeneic cell linesthat are mismatched to the patients' haplotype. Also described aremismatched allogeneic prostate cell lines transfected with GM-CSF.

Despite this intensive work in a crucial field of medical science,successful and reproducible eradication or inhibition of cancer growthremains elusive. Any new material or procedure that can address and atleast partially overcome the limitations inherent in the use of cellbased vaccines would provide very important benefits for treatment ofthis disease. These needs are satisfied by the present invention.

SUMMARY OF THE INVENTION

Embodiments of the invention alleviate the problems in the fieldsummarized above in several ways. One embodiment provides an allogeneicimmunotherapy vaccine for the treatment of prostate cancer in a patient,comprising an adjuvant, cells from a first allogeneic normal prostatecell line, cells from a second allogeneic cell line obtained from aprimary prostate cancer biopsy, and cells from a third allogeneic cellline obtained from a metastasis of prostate cancer.

Another embodiment provides an allogeneic immunotherapy vaccine for thetreatment of prostate cancer in a patient, comprising an adjuvant,allogeneic cells from a first normal prostate cell line, allogeneiccells from a second immortalized cell line obtained from a prostatecancer biopsy, and allogeneic cells from a third immortalized lineobtained from a prostate cancer biopsy, wherein cells of the secondimmortalized cell line express normal levels of neutral endopeptidasebut low levels of endothelin converting enzyme and cells of the thirdimmortalized cell line express low levels of both neutral endopeptidaseand endothelin converting enzyme.

Yet another embodiment provides an allogeneic immunotherapy vaccine forthe treatment of prostate cancer in a patient, comprising an adjuvantand a combined vaccine ONY-P1, wherein the ONY-P1 comprises allogeneiccells from a first normal prostate cell line, allogeneic cells from asecond immortalized cell line obtained from a prostate cancer metastasisbiopsy, and allogeneic cells from a third immortalized line obtainedfrom a prostate cancer metastasis biopsy.

One aspect of the invention provides methods of inhibiting progressionof prostate cancer in a patient, comprising administering the patient aneffective amount of an allogeneic immunotherapy vaccine, wherein thevaccine comprises an adjuvant and a combined vaccine ONY-P1, wherein theONY-P1 comprises allogeneic cells from a first normal prostate cellline, allogeneic cells from a second immortalized cell line obtainedfrom a prostate cancer biopsy, and allogeneic cells from a thirdimmortalized line obtained from a prostate cancer biopsy. In anotheraspect, the invention provides methods of increasing PSA doubling times(PSADT) in a prostate cancer patient by administering the patient aneffective amount of the allogeneic immunotherapy vaccine.

Another aspect of the invention provides methods of inhibitingprogression of prostate cancer in a patient, comprising administeringthe patient an effective amount of an allogeneic immunotherapy vaccine,wherein the vaccine comprises an adjuvant, cells from a first allogeneicnormal prostate cell line, cells from a second allogeneic cell lineobtained from a primary prostate cancer biopsy, and cells from a thirdallogeneic cell line obtained from a metastasis of prostate cancer. Inanother aspect, the invention provides methods of increasing PSADT in aprostate cancer patient by administering the patient an effective amountof the allogeneic immunotherapy vaccine.

Another aspect of the invention provides methods of inhibitingprogression of prostate cancer in a patient, comprising administeringthe patient an effective amount of an allogeneic immunotherapy vaccine,wherein the vaccine comprises an adjuvant, cells from a first allogeneicnormal prostate cell line, cells from a second allogeneic cell lineobtained from a primary prostate cancer biopsy, and cells from a thirdallogeneic cell line obtained from a metastasis of prostate cancer,wherein the cells of the second allogeneic cell line exhibit tumourassociated glycoprotein related to sialylated Tn antigen. In anotheraspect, the invention provides methods of increasing PSADT in a prostatecancer patient by administering the patient an effective amount of theallogeneic immunotherapy vaccine.

In another aspect, the invention provides methods of inhibitingprogression of prostate cancer in a patient, comprising administeringthe patient an effective amount of an allogeneic immunotherapy vaccine,wherein the vaccine comprises an adjuvant, allogeneic cells from a firstnormal prostate cell line, allogeneic cells from a second immortalizedcell line obtained from a prostate cancer biopsy, and allogeneic cellsfrom a third immortalized line obtained from a prostate cancer biopsy.In another aspect, the invention provides methods of increasing PSADT ina prostate cancer patient by administering the patient an effectiveamount of the allogeneic immunotherapy vaccine.

Yet in another aspect, the invention provides methods of inhibitingprogression of prostate cancer that has metastasised to a tissueselected from the group consisting of bone, lymph node, brain and liverin a patient, comprising administering the patient an effective amountof an allogeneic immunotherapy vaccine, wherein the vaccine comprises anadjuvant, allogeneic cells from a first normal prostate cell line,allogeneic cells from a second immortalized cell line obtained from aprostate cancer biopsy, and allogeneic cells from a third immortalizedline obtained from a prostate cancer that has metastasised. Yet inanother aspect, the invention provides methods of increasing PSADT in aprostate cancer patient by administering the patient an effective amountof the allogeneic immunotherapy vaccine.

Other embodiments will be appreciated by a skilled artisan upon readingthe specification. Many of these embodiments concern selected cell linesused in allogeneic immunotherapy agents for the treatment of cancer.These types of vaccine provided unexpected favourable clinical outcomes.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a plot of proliferation index for various cell lysates.

FIGS. 2A-2B, 2C-2D, and 2E-2F show Western Blot analyses of serum frompatients 115, 304, and 402, respectively.

FIGS. 3A-3L show antibody titres of serum from three patients.

FIGS. 4A, 4B, and 4C show PSA values for three patients.

FIG. 5 shows survival curves for C57 mice immunized with normalmelanocytes.

FIG. 6 shows a PSA level in a non-responding patient increasinglogarithmically.

FIG. 7 shows reduction in the rate of PSA increase for a patient.

FIG. 8 shows reduction in the rate of PSA increase for a patient.

FIG. 9 shows PSA doubling time results for 15 patients.

FIG. 10 shows the ratio of PSA doubling time for 15 patients.

FIG. 11 shows the effect of ONY-P1 treatment on PSA doubling times(PSADT) of patients in cohort group 1 (cohort 1).

FIG. 12 shows the effect of ONY-P1 treatment on PSA doubling times(PSADT) of patients in cohort group 2 (cohort 2).

FIG. 13 depicts assessed time to disease progression (TTP) in patientsof cohort group 1 (cohort 1).

FIG. 14 depicts assessed TTP in patients of cohort group 2 (cohort 2).

DETAILED DESCRIPTION OF THE INVENTION

Generally, cell-based cancer vaccines until now have a common feature ofemploying cells that contain at least some TSAs and/or TAAs that areshared with antigens present in a patient's tumour. In each case, tumourcells are utilized as the starting point on the premise that only tumourcells will contain TSAs or TAAs of relevance, and the tissue origins ofthe cells are matched to the tumour site in patients.

In contrast to this expectation in the field, some embodiments of theinvention utilize immortalized ‘normal,’ non-malignant cells as a basisof allogeneic cell cancer vaccines. Normal cells are not expected toposses TSAs or relevant concentrations of TAAs. Hence it was surprisingthat normal cells, and particularly combinations of normal cells withcells derived from tumour biopsies as described herein are effective asanti-cancer vaccines. The approach is general and can be adapted to anymammalian tumour by the use of immortalized normal cells derived fromthe same particular tissue as the tumour intended to be treated.Immortalized normal cells can be prepared by those skilled in the artusing published methodologies, or they can be sourced from cell bankssuch as ATCC or ECACC, or they are available from several researchgroups in the field.

A prostate cancer vaccine, for example may include one or a combinationof different immortalized normal cell lines derived from the prostateand can be prepared using methods reviewed and cited in Rhim, J. S, andKung, H-F., 1997 Critical Reviews in Oncogenesis 8(4):305-328 orselected from PNT1A (ECACC Ref. No. 95012614), PNT2 (ECACC Ref. No.95012613) or PZ-HPV-7 (ATCC No. CRL-2221). In an embodiment, a clonalderivative of PNT-2, named OnyCap-23 (ECACC Ref. No. 00032801) desirablyis combined with cells obtained from primary or metastatic cancerbiopsies. Accordingly, a further embodiment is the addition of TSAsand/or TAAs by combining one or more immortalized normal cell line(s)such as OnyCap-23 with one, two three or more different cell linesderived from primary and/or metastatic cancer biopsies. In anembodiment, prostate cancer cells from at least one cell line derivedfrom a metastasis biopsy from lymph node, bone, brain or liver tissueare combined with at least one cell line derived from a biopsy ofunmetastasised tissue from a prostate. In a particularly favourableembodiment, the metastasised sample derived cell line is LnCaP (ATCC No.CRL-1740) and the cell line from a primary prostate cancer biopsy isP4E6 (ECACC Ref. No. 04071601. Also, see Maitland et al., RadiationResearch 155: 133-142 (2001). The compositions of these cell lines mayfurther include cells or cell lines.

In another embodiment pursuant to the strategy of combining cells from anormal tissue cell line with other cells from at least two identifiablestages of cancer progression, cells from a normal prostate cell line arecombined with at least one cell line representative of non-metastasisedcells and at least one cell line representative of metastasised cells.

In an embodiment preferably cells from the LnCaP and P4E6 cell lines arecombined with cells from a normal prostate cell line (such asOnyCap-23). The LnCaP and P4E6 cells in such formulations may bereplaced or supplemented with cells from other cell lines that arederived from lymph node metastases or primary prostate cancersrespectively may replace the LnCaP cells. Other cell linesrepresentative of normal prostate cells may replace OnyCap-23, such as,for example, PNT-2 cells (see Maitland et al., Radiation Research 155:133-142 (2001).

In yet another embodiment, a prostate cell line obtained from a tumourbiopsy that exhibits high neutral endopeptidase-24.11 activity and lowendothelin-converting enzyme activity (for example, as determined usingthe methods of Usmani et al., the relevant passages of which areparticularly incorporated by reference) may replace the LnCaP cells. Inyet another embodiment, a prostate cell line obtained from a tumourbiopsy that exhibits low levels of both neutral endopeptidase-24.11 andendothelin-converting enzyme may replace the P4E6 cells. In anotherembodiment, other cell lines representative of normal prostate cells mayreplace OnyCap-23, such as, for example, PNT-2 cells.

In yet another embodiment, a vaccine comprises allogeneic cells from afirst normal prostate cell line, allogeneic cells from a secondimmortalized cell line obtained from a prostate cancer biopsy thatexpress normal levels of neutral endopeptidase but low levels ofendothelin converting enzyme, and allogeneic cells from a thirdimmortalized line obtained from a prostate cancer biopsy that expresslow levels of both neutral endopeptidase and endothelin convertingenzyme. In this instance, the presence of tumour associated glycoproteinrelated to sialylated Tn antigen, as described by the work of Brenner etal. (see J. Urology 153: 1575-1579 1995) may be used to select a cellline obtained from a the primary prostate cancer. The differentialpresentation of neutral endopeptidase-24.11 and endothelin convertingenzyme as described by Usmani et al. in Clin. Science 103 (supp 48):3145-3175 can be used for this embodiment.

All the cell lines described herein will show good growth in large scalecell culture and sufficient characterization to allow for qualitycontrol and reproducible production.

Preferably, the cell lines are lethally irradiated utilizing gammairradiation at 20-400 Gy to ensure that they are replication incompetentprior to use in the mammal or human.

The cell lines and combinations referenced herein preferably are frozen,freeze dried or otherwise stabilized to allow their transportation andstorage. Accordingly, in a further embodiment a combination of cellsreferenced herein may be formulated with a cryoprotectant solution.Suitable cryoprotectant solutions may include but are not limited to,10-30% v/v aqueous glycerol solution, 5-20% v/v dimethyl sulphoxide or5-20% w/v human serum albumin may be used either as singlecryoprotectants or in combination.

Cells obtained from the cell lines may be mixed in any convenientproportion. Preferably, no cell type is present in the mixture more than20 fold (measured using total amounts of DNA) more than any other celltype. More preferably the ratio is less than 10, 5 or 3 fold.

Yet a further embodiment is the use of a cell line combination with anon-specific immune stimulant such as BCG, M. Vaccae, a Mycobacterium,Tetanus toxoid, Diphtheria toxoid, Bordetella Pertussis, interleukin 2,interleukin 12, interleukin 4, interleukin 7, Complete Freund'sAdjuvant, Incomplete Freund's Adjuvant or another known non-specificagent. Such general immune stimulants advantageously can enhance immunestatus whilst the combinations of cell lines, contribute to immuneenhancement via haplotype mismatch and, at the same time, target animmune response to a plethora of TAA and TSA due to their heterogeneity.

The invention is further described by the following examples, which donot limit the invention in any manner.

EXAMPLES Example 1 Growth, Irradiation, Formulation and Storage of Cells

An immortalised cell line derived from normal prostate tissue namelyPNT2 was grown in roller bottle culture in RPMI 1640 media supplementedwith 2 mM L-glutamine and 5% fetal calf serum (FCS) following recoveryfrom liquid nitrogen stocks. Following expansion in T175 static flasksthe cells were seeded into roller bottles with a growth surface area of850 cm² at 1-20×10⁷ cells per roller bottle.

An immortalized cell line derived from primary prostate tissue namelyNIH1542-CP3TX (ATCC No. CRL-12037) was grown in roller bottle culture inKSFM media supplemented with 25 μg/ml bovine pituitary extract, 5 ng/mlof epidermal growth factor, 2 mM L-glutamine, 10 mM HEPES buffer and 5%fetal calf serum (FCS) (hereinafter called “modified KSFM”) followingrecovery from liquid nitrogen stocks. Following expansion in T175 staticflasks the cells were seeded into roller bottles with a growth surfacearea of 1,700 cm² at 2-5×10⁷ cells per roller bottle.

Two secondary derived cell lines, LnCap (ATCC No. CRL-1740) and Du145(HTB-81), obtained from ATCC were used. LnCap was grown in large surfacearea static flasks in RPMI media supplemented with 10% FCS and 2 mML-glutamine following seeding at 1-10×10⁶ cells per vessel and thengrown to near confluence. Du-145 was expanded from frozen stocks instatic flasks and then seeded into 850 cm² roller bottles at 1-20×10⁷cells per bottle and grown to confluence in DMEM medium containing 10%FCS and 2 mM L-glutamine. All cell lines were harvested utilisingtrypsin at 1× normal concentration. Following extensive washing in DMEMthe cells were re-suspended at a concentration of 5-40×10⁶ cells/ml andirradiated at 50-300 Gy using a Co⁶⁰ source. Following irradiation thecells were formulated in cryopreservation solution composing of 10%DMSO, 8% human serum albumin in phosphate buffered saline, and frozen ata cell concentration of 5-150×10⁶ cells/ml, in liquid nitrogen untilrequired for use.

Vaccination Schedule

Prostate cancer patients were selected on the basis of being refractoryto hormone therapy with a serum PSA level of at least 30 ng/ml.

Cell Lines Administered Dose Trial Arm A Trial Arm B Trial Arm C 1, 2and 3 PNT2 Du145 LnCap 4 and subsequent PNT2/Du145/ PNT2/Du145/PNT2/NIH1542/ NIH1542 LnCap LnCap

The cells were warmed gently in a water bath at 37° C. and admixed withmycobacterial adjuvant prior to injection into patients. Injections weremade intra-dermally at four injection sites into draining lymph nodebasins. The minimum interval between doses was two weeks, and most ofthe doses were given at intervals of four weeks. Prior to the firstdose, and prior to some subsequent doses, the patients were tested fordelayed-type hypersensitivity (DTH) against the four cell lines listedin the vaccination schedule above (all tests involved 0.8×10⁶ cells withno adjuvant).

Immunological responses were analysed. T-Cell proliferation responseswere determined as follows. To evaluate the expansion of T-cellpopulations that recognize antigens of the vaccinating cell lines, a Tcell proliferation assay was used that employed stimulation with lysatesfrom the prostate cell lines. Whole blood was extracted at each visit tothe clinic and used in a BrdU (bromodeoxyuridine) based proliferationassay as described below:

Patient BrdU proliferation method Reagents RPMI Life Technologies,Paisley, Scotland BrdU Sigma Chemical Co, Poole, Dorset PharMlyse 35221EPharmingen, Oxford UK Cytofix/Cytoperm 2090KZ ″ Perm/Wash buffer (×10)2091KZ ″ FITC Anti-BrdU/Dnase 340649 Becton Dickinson PerCP Anti-CD3347344 ″ Pe Anti-CD4 30155X Pharmingen Pe Anti-CD8 30325X ″ FITC mu-IgG1349041 Becton Dickinson PerCP IgG1 349044 ″ PE IgG1 340013 ″

In this method, 1 ml blood is diluted with 9 ml RPMI+2 mM L-gln+PS+50 μM2-Me. Serum should not be added. The blood is left overnight at 37° C.The following morning, 450 μl of diluted blood were aliquoted into wellsof a 48-well plate and 50 μl of stimulator lysate added. The lysate wasmade by freeze-thawing tumour cells (2×10⁶ cell equivalents/ml) ×3 inliquid nitrogen and then stored aliquots frozen until required. Cellsare cultured at 37° C. for 5 days. On the evening of day 5 50 μl BrdU at30 μg/ml are added. One hundred μl of each sample are aliquoted eachinto cells of a 96-well round-bottomed plate. Each plate is spun andsupernatant discarded. Red cells are lysed using 100 μl Pharmlyse for 5minutes at room temperature, and then washed ×2 with 50 μl of Cytofix.The samples are spun and supernatant removed by flicking. Then the cellsare permeabilized with 100 μl Perm wash for 10 mins at RT. Thirtymicroliters of antibody mix are added, that comprise antibodies atcorrect dilution made up to volume with Perm-wash. The mixtures areincubated for 30 mins in the dark at room temperature. This is followedby wash ×1 and resuspension in 100 μl 2% paraformaldehyde. This is addedto 400 μl FACSFlow in cluster tubes ready for analysis. Analysis iscarried out with a FACScan, and storing 3000 gated CD3 events.

The set ups of microtiter plate conditions are shown in Table 1 andTable 2.

TABLE 1 6-well plate for stimulation. Nil ConA 1542 LnCap Du145 Pnt2 PBL1 PBL 2 PBL 3 PBL 4 PBL 5 PBL 6

The results for the proliferation assays are shown in FIG. 1, wherein aproliferation index for either CD4 or CD8 positive T-cells are plottedagainst the various cell lysates. The proliferation indexes are derivedby dividing through the percentage of T-cells proliferating by theno-lysate control.

Results are shown for three patients (numbers 112, 307 and 406). Resultsare given for four cell lysates namely, NIH1542, LnCap, DU-145 andPNT-2. Overall, 50% of patients treated mount a specific proliferativeresponse to at least one of the cell lines.

Western blots using patient serum were carried out. Standardized celllysates were prepared for a number of prostate cell lines to enableloading of similar quantities of protein on a denaturing SDS PAGE gelfor Western blot analysis. Each blot was loaded with molecular weightmarkers, and equal amounts of protein derived from cell lysates ofNIH1542, LnCap, DU-145 and PNT-2. The blot then was probed with serumfrom patients derived from pre-vaccination and following 16 weeksvaccination (four to six doses).

TABLE 2 96-well plate for antibody staining. PBL 1 PBL 2 PBL 3 PBL 4 PBL5 PBL 6 Nil A 15 D Nil A 15 D Nil A 15 D Nil A 15 D Nil A 15 D Nil A 15D Nil D 15 E Nil D 15 E Nil D 15 E Nil D 15 E Nil D 15 E Nil D 15 E NilE Ln D Nil E Ln D Nil E Ln D Nil E Ln D Nil E Ln D Nil E Ln D Con D Ln ECon D Ln E Con D Ln E Con D Ln E Con D Ln E Con D Ln E Con E Du D Con EDu D Con E Du D Con D Du D Con E Du D Con E Du D Du E Du E Du E Du E DuE Du E Pn D Pn D Pn D Pn D Pn D Pn D Pn E Pn E Pn E Pn E Pn E Pn E TableLegend: A: IgG1-FITC (5 μl) IgG1-PE (5 μl) IgG1-PerCP (5 μl) 15 μlMoAb +15 μl) D: BrdU-FITC (5 μl) CD4-PE (5 μl) CD3-PerCP (5 μl) 15 μlMoAb + 15μl E: BrdU-FITC (5 μl) CD8-PE (5 μl) CD3-PerCP (5 μl) 15 μlMoAb + 15 μl15: NIH1542-CP3TX Ln: LnCap D: Du1145 Pn: PNT2 Con: ConA lectin(positive control) Nil: No stimulation

In this method, of sample preparation from prostate tumour lines, cellpellets are washed 3 times in PBS, and then re-suspended at 1×10⁷cells/ml of lysis buffer. The re-suspended cells are passed through 5cycles of rapid freeze thaw lysis in a liquid nitrogen/water bath. Thecells then are centrifuged at 1500 rpm for 5 minutes to remove celldebris, and ultracentrifuged at 20,000 rpm for 30 min to remove membranecontaminants. These are aliquoted at 200 μl and stored at −80° C. Gelelectrophoresis is carried out by mixing lysates 1:1 with Laemellisample buffer and boiling for 5 minutes. Then, 20 μg samples are loadedinto 4-20% gradient gel wells. The sample gels are electrophoresed inBjerrum and Schafer-Nielson transfer buffer (with SDS) at 200 V for 35minutes.

Western transfer methods were carried out by equilibrating gels,nitrocellulose membranes and blotting paper in transfer buffer for 15minutes. Western blot data from serum of patients 115, 307, and 406 arepresented as FIGS. 2A-2B, 2C-2D, and 2E-2F, respectively. Thengel-nitrocellulose sandwiches are arranged on anodes of semi-dryelectrophoretic transfer cells made from 2 sheets of blotting paper,nitrocellulose membrane, gel, and 2 sheets of blotting paper. A cathodeis applied and sandwiches exposed to 25 V for 90 minutes. Immunologicaldetection of proteins was carried out by blocking nitrocellulosemembranes overnight at 4° C. with 5% Marvel in PBS/0/05% Tween 20. Themembranes were rinsed twice in PBS/0.05% Tween 20, then wash for 20 minand 2×5 min at RT on a shaking platform. The membranes were thenincubated in 1:20 dilution of clarified patient plasma for 120 min at RTon a shaking platform. This was followed by a wash as above with anadditional 5 min final wash. The membranes were then incubated in 1:250dilution of biotin anti-human IgG of IgM for 90 min at RT on a shakingplatform, then washed as above with an additional 5 min final wash. Thenthe membranes are incubated in 1:1000 dilution ofstreptavidin-horseradish peroxidase conjugate for 60 min at RT on ashaking platform, and washed as above. The membranes are then incubatedin Diaminobenzidine peroxidase substrate for 5 min to allow colourdevelopment. The reaction is stopped by rinsing the membranes withwater.

FIGS. 3A, 3B, and 3C show 3 results obtained from three patients (112,305, and 402, respectively). These results show clearly that vaccinationover a 16 week period, using four to six doses, can cause an increase inantibody titre against cell line lysates as well as cross reactivityagainst lysates not received in this vaccination regime (other than DTHtesting).

Antibody titres were determined by coating ELISA plates withstandardised cell line lysates and by dilution studies on serum fromvaccinated patients. The Elisa method utilized anti-lysate IgG. Plateswere coated with 50 μl/well lysates at 10 μg/ml according to dilutionsin the following Table 3.

TABLE 3 Dilution Table. Lysate Protein conc Coating conc Amount/mlAmount PNT2 2.5 mg/ml 10 μg/ml 3.89 μl 19.4 μl 1542 4.8 mg/ml 10 μg/ml2.07 μl 10.3 μl Du145 2.4 mg/ml 10 μg/ml 4.17 μl 20.8 μl LnCap 2.4 mg/ml10 μg/ml 4.12 μl 20.6 μl

Each sample was covered and incubate overnight at 4° C., followed bywash ×2 with PBS-Tween. Each plate was pounded on paper towels to dryand then samples blocked with PBS/10% FCS (100-μL/well). These werecovered and incubate at room temperature (RT) for 1 hour (minimum) andthen wash ×2 with PBS-Tween. Then 100 μl PBS-10% FCS were added tosamples in rows 2-8, 200 μl plasma samples (diluted 1 in 100 in PBS-10%FCS that is, 10 μl plasma added to 990 μls PBS-10% FCS) to row 1 andserial 100 μl dilutions made down the plate below the row. The extra 100μl from bottom wells were discarded and each plate covered and incubatedin a refrigerator overnight.

Biotinylated antibody solution (Pharmingen; IgG 34162D) was diluted andadded ie. final concentration 1 mg/ml (ie. 20 ml in 10 mls). The sampleswere covered and incubated at RT for 45 minutes and washed ×6 as above.A dilute streptavidin-HRP conjugate obtained from Pharmingen, (13047E 0)was diluted 1:1000 (ie. 10 ml->10 mls) and added to 100 ul/well. Thesamples were incubated at 30 min at RT and then wash ×8. Then 100 μlsubstrate solution was added to each well and signal allowed to developfor 10-80 min at RT. The colour reaction was stopped by adding 100 ul 1MH₂SO₄ per well and the optical densities determined at 405 nm.

Results obtained indicated that, antibody titres at baseline (0), 4weeks, 8 weeks and 16 weeks for the 3 patients (112, 305 and 402)increase. The data show that after vaccination with at least four doses,patients exhibit increased antibody titre against cell line lysates andalso cross-reactivity against cell lines not received in thisvaccination regime (except as DTH doses).

PSA levels were evaluated for patients receiving the vaccine at entryinto the trial and throughout the course of vaccination, using routinelyused clinical kits. The PSA values for three patients (110, 303, and404) are shown in FIGS. 4A, 4B, and 4C, respectively (vertical axis isserum PSA in ng/ml; horizontal axis is time, with the first time pointrepresenting the initiation of the vaccination programme) and portray adrop or partial stabilization of the PSA values, which in this group ofpatients normally continues to rise, often exponentially. The result forpatient 110 is somewhat confounded by the radiotherapy treatment toalleviate bone pain, although the PSA level had dropped prior toradiotherapy.

Example 2 Use of a Normal Melanocyte in a Murine Melanoma ProtectionModel

A normal melanocyte cell line was used in a vaccination protection modelof murine melanoma utilising the B16.F10 as the challenge dose. The C57mice received two vaccinations of either PBS, 5×10⁶ irradiated K1735allogeneic melanoma cells or 5×10⁶ irradiated Melan P1 autologous normalmelanocyte cells on days −14 and −7. Challenge on day 0 was with 1×10⁴B16.F10 cells and tumor volume measured every three days from day 10onwards. Animals were sacrificed when the tumor had grown to 1.5×15 cmmeasured across the maximum dimensions of the tumor. It was found thatthat vaccination with Melan1P cells offer some level of protectionagainst this particularly aggressive murine tumour, as seen in FIG. 5.

Example 3 Phase I/II Study

A phase I/II study was carried out with three types of allogeneic cellsrepresenting a normal prostate cell line, a prostate tumour derived cellline and a metastasised tumour cell line. A combined cell vaccine wasgiven to patients having hormone refractory prostate cancer and safety,tolerability and efficacy, as measured by effect on survival and qualityof life determined. The following criteria were used for inclusion ofpatients: patients of any age with histologically confirmed prostatecancer; patients with hormonal refractory disease following optimalfirst line LHRH treatment, or high dose bicalutamide (150 mg per day),or orchidectomy; progressive disease indicated by a rise in serum PSA onat least 2 successive occasions separated by at least 4 weeks; serum PSAlevel of at least 2 ng/ml at Week −2; WHO Performance Status of 0-2 atWeek −4; a life expectancy of at least 6 months; the ability of thepatient to read and understand the patient information leaflet and togive written informed consent; the willingness and ability of thepatient to attend the hospital for all treatments and assessments;adequate bone marrow function (WBC>3500/mm³, haemoglobin>9 g/dl,platelet count>100,000/mm³ at Week 0); adequate renal function at Week 0(serum creatinine<2.0 mg/dl); adequate hepatic function at Week 0 (<2times upper limit of normal, ALT 52 U/l, AST 40 U/l); adequate responseto DTH testing with the specified intra-dermal antigens at Week 0; andnormal 24 hour Urinary Cortisol at Week −2 (60-240 nmol/24 hr).

The following criteria were used for removing a patient from the study.A patient could withdraw at any time without reason. The investigatorcould withdraw a patient if it is in the best interest of the patient.Commencement on any other investigational agent, radiotherapy,chemotherapy or corticosteroids (for example, in spinal cordcompression) or surgical intervention was another criteria. Protocolviolation by investigator or patient that in the opinion of theSponsor's medical expert would interfere with the study was another.Unacceptable toxicity, disease progression as measured by appearance ofnew metastatic lesions confirmed by radiological investigations, andsymptomatic disease progression also could prompt withdrawal. Patientswho withdrew before completion of 6 months treatment with ONY-P1 werereplaced unless withdrawal was due to disease progression orunacceptable toxicity.

An open label safety, tolerability and efficacy trial was carried out asfollows. A combined vaccine “ONY-P1” was used in a translucent plasticCryo Sleeve that contained three Greiner Cryo.S vials, each containing8×10⁶ irradiated cells. Each vial contained one of the following celllines: Vial 1: LnCaP (Code CT3); Vial 2: P4E6 (Code CT4); Vial 3:OnyCap-23 (Code CT1). The vials were stored in the vapour phase ofliquid nitrogen at −178° C. and transported to an investigational siteon the day of administration in a Dewar Flask that contains liquidnitrogen. Cells were suspended in Hanks Balanced Salts Solution plus 2%foetal calf serum plus 8% dimethyl sulfoxide. BCG was obtained by Onyvax(OncoTICE, N.V. Organon, Kloosterstraat 6, PO Box 20, 5340 BH Oss, TheNetherlands, PL 05003/0046) and each dose contained 0.6-2.4×10⁶ CFU (therange stems from the product label). The product was diluted in salinefor injection to yield the specified dose in 0.1 ml. Each injectionsuspension contained the contents of three complete vials (one of eachcell type). The total volume (made up with the requisite volume ofsaline for injection) was 1 ml, given as 8×0.125 ml intradermalinjections, with two injections into each draining lymph node basin. TheBCG-adjuvanted doses were given at weeks 0 and 2; cells alone are givenat weeks 4, 8, and at 4-weekly intervals up to and including week 48 (14doses in total).

TABLE 4 Treatment Schedule. Week Number Activity −4 −2 0 2 4 8 12 13 1415 16 20 24 28 32 PSA (10 ml)¹ Y Y Y Y Y Y Y Y Y Y Y Y Chemistry (10ml)² Y Y Y Y Y Y Y Y Y Y Y Haematology (5 ml)³ Y Y Y Y Y Y Y Y Y Y YImmunology profile Y Y Y Y Y Y Y Y Y Y Y DTH Antigens Y Y DTH Cell LinesY Y 24 h urinary cortisol Y Physical exam⁵ Y Y Y Y Chest X-ray Y Y YBone scan Y Y CT abdomen/pelvis Y Y EORTC QLQ-30 Y Y Skin punch biopsy YY BCG Y Y ONY-P1 Y Y Y Y Y Y Y Y Y Y Week Number Activity 33 34 35 36 4044 48 49 50 51 52 PSA (10 ml)¹ Y Y Y Y Y Chemistry (10 ml)² Y Y Y Y YHaematology (5 ml)³ Y Y Y Y Y Immunology profile Y Y Y Y Y Y Y Y Y DTHAntigens Y Y DTH Cell Lines Y 24 h urinary cortisol Physical exam⁵ Y YChest X-ray Y Y Bone scan Y CT abdomen/pelvis Y EORTC QLQ-30 Y Skinpunch biopsy Y BCG ONY-P1 Y Y Y Y ¹Sample to be taken in Na-Heparinvacutainer. If sample taken on the same occasion as for Chemistry, thenone 10 ml sample will suffice for both. ²Sample to be taken inNa-Heparin vacutainer. If sample taken on the same occasion as forChemistry, then one 10 ml sample will suffice for both. ³Sample to betaken into EDTA. ⁴Samples to be taken in Na-Heparin. 10 ml volume to betaken on all occasions unless written request for 50 ml made by Sponsor.⁵Including Physical/clinical assessment, vital signs and InternationalProstate Symptom Score.

The study was an open label design in a maximum of 48 evaluable patientssplit into two cohort groups. The first cohort group (cohort 1) wascomposed of 28 patients without bone metastases and the second cohortgroup (cohort 2) was composed of 20 patients with bone metastases. Thetotal treatment period for each individual patient was 12 months. Thethree-stage study was as follows: i.) stage one, a pre treatment phaseand an initial treatment phase lasting four weeks in which patientsreceive ONY-P1 plus BCG; ii) stage two, which lasted 48 weeks whereinpatients were treated once a month with ONY-P1 alone; and iii) stagethree, a follow up of all patients for 12 months following completion oftreatment.

The treatment-assay schedule shown in Table 4 above was carried out.

In an initial analysis of the data, the first 15 patients who receivedmore than 4 months of treatment were reviewed in great detail Of the 15,5 showed statistically significant PSA velocity reductions. It wasconcluded that there were no safety issues, 5 of the 15 showedstabilization of PSA titres that correlated with their immunologicalprofile (Th1/Th2).

PSA data from the clinical trial are presented by plotting each patienton a log scale. See FIGS. 6 through 8. Data taken prior to vaccinationare shown in grey lines, which corresponds to the left side of the plotuntil the sustained increase in FIG. 6, and the first third of the plotsof FIGS. 7 and 8 prior to levelling off. Data taken during vaccinationis shown as later data points which are more leveled off. In hormonerefractory prostate cancer the PSA level increases logarithmically untildeath. Certain existing therapies do show effects on PSA velocity, butthese have been transient and not associated with effects on patientsurvival. At this disease stage, the PSA velocity either remainsconstant, or, as shown by patient 6 (non-responder) below, increases.

FIGS. 6 through 8 show PSA levels in individual patients, whichincreased logarithmically. A control, or non-responder as represented bypatient results shown in FIG. 6 shows a PSA level that increaseslogarithmically until death. The y axis depicts ln PSA concentration andthe x axis depicts time over a year period.

Some typical existing therapies show effects on the “PSA velocity” asseen in the dip in the plot of FIG. 6, but such effects generally aretransient and not associated with effects on patient survival. Incontrast, five of the first 15 patients analysed, sustainedstatistically significant reductions in PSA velocity (for example,patients 1 and 14), the data of which are shown in FIGS. 7 and 8. Thedata to the left of the inflection mark show velocity prior to treatmentand the data points to the right show the lower velocity aftertreatment. The straight lines from left to right indicate the linearregression of the data before and after treatment, respectively. Moreimportantly, a decrease in velocity of PSA was seen equivalent to anincrease in PSA doubling time, as summarized in FIGS. 9 and 10. FIG. 9provides representative data from 15 patients and FIG. 10 depicts theratio of PSA doubling times before and after treatment.

ONY-P1 clinical studies and PSA data. As described above, the studyincluded 48 hormone-resistant prostate cancer (HRPC) patients split intotwo cohort groups. Vaccine composition and dosages also are described inExample 3. Cohort group 1 was composed of 28 patients with no bonemetastases and cohort group 2 was composed of 20 patients withasymptomatic bone metastases. Twenty-six of 28 patients of the cohortgroup 1 and 13 of 20 patients from the cohort group 2 were treated. Theremaining patients were excluded from the study for non-compliance withthe study protocol. The total treatment period for each individualpatient was 12 months with intradermal injections. The three-stage studywas as follows: i) stage one, a pre treatment phase and an initialtreatment phase lasting up to two weeks in which patients receive ONY-P1plus BCG; ii) stage two, which lasted up to 4-48 weeks wherein patientswere treated once a month with ONY-P1 alone; and iii) stage three, afollow up of all patients for 12 months following completion oftreatment.

FIG. 11 shows the effect of ONY-P1 treatment on PSA doubling times(PSADT) of patients in cohort group 1. All pre-treatment PSA data andthe PSA data during the treatment were collated in a database. Naturallog(ln) PSA was plotted against time and the gradients of the plotscorresponded to the PSA velocity (PSAV). PSADT was derived by theformula ln(2)/PSAV. The ratio of “on-treatment PSADT” to “pre-treatmentPSADT” is shown in the FIG. 11. Ratios greater than 1 indicate anincrease in PSADT induced by ONY-P1 therapy, and vice versa. It is knownin the art that PSADT naturally decreases as HRPC progresses andgenerally, a spontaneous increase in PSADT is not observed. In FIG. 11,stars indicate patients in which the increased PSADT is statisticallysignificant (p<0.05), as determined by multiple regression using astandard analysis on the Graph Pad Prism 3 computer programme.

Referring to FIG. 11, 11 out of the 26 treated patients of cohort group1 exhibited statistically-significant increases in PSADT. Bymathematical definition, this also means that the same 11 patientsshowed a statistically-significant reduction in PSAV. The mean increasein PSADT in the eleven patients was two-fold.

An equivalent analysis also was performed for patients in cohort group 2and the results are depicted in FIG. 12. Five of the 13 treated patientsin cohort group 2 showed a statistically-significant increase in PSADTas determined by the Graph Pad Prism 3 analysis (see FIG. 12). The meanincrease in PSADT in the five patients was over three-fold.

Time to disease progression (TTP) is an acceptable clinical end point inHRPC studies. TTP also is a pivotal trial end point in current Phase IIIclinical trials. Disease progression in patients in both cohort groupswere assessed and depicted in FIGS. 13 and 14.

The median TTP in cohort group 1 is 58 weeks (See FIG. 13) and forcohort group 2 it is 23 weeks (See FIG. 14), as determined by the timepoint at which the percent progression-free patients reaches 50%.

The findings of this study are significant for several reasons, forexample,

-   -   1. The effects of vaccine therapy on PSADT were not known until        this study. The effects were maintained over a prolonged period        of time. PSAV data show reduced velocities for periods of 6 to        18 months in about 40% of the patients (See FIGS. 11 and 12).        According to recent studies, PSADT and PSAV are predictors of        prostate cancer disease progression and survival in HRPC (see        Nelson et al., 2004 ASCO Annual Meeting, Abstract No. 4554 and        D'Amico et al., 2004 ASCO Annual Meeting, Abstract No. 4506).    -   2. Median TTP in cohort group 1, using standard criteria, is        observed to be 58 weeks (see FIG. 13). Whereas, median TTP        generally for patients with no bone metasteses is known in the        art to be about 26-28 weeks (see Atlas of Clinical Urology, Ed.        Peter T. Scardino MD (1999, 2002); Carducci et al., J Clin        Oncology (2003) 21:697-689; Small et al., J Clin Oncology (2000)        18:3894-3903).    -   3. Median TTP in patients with asymptomatic bone metasteses is        known in the art to be about 9 weeks (see Proc. Am Asso Cancer        Res (July 2003) vol. 44, 2nd ed., Abstract 5396). In contrast,        Median TTP in cohort group 2 is found to be 23 weeks in this        study. And    -   4. The patient population studies in ONY-P1 trial was composed        of “all comers”, and was not selected or subdivided on the basis        of disease severity. For example, patient population was        selected or subdivided using the Gleason scores to differentiate        between grades of disease by other investigators (see Proc. Am        Asso Cancer Res (July 2003) vol. 44, 2nd ed., Abstract 5396).

The apparent efficacy of ONY-P1 appeared to be surprising while comparedto previous trials using other vaccines; and Eaton et al., BJUInternational (2002) 89:19-26), wherein it was possible to measureeffects in patients with bone metastases which are comparable to thoseobserved in patients without bone disease, i.e. at an earlier stage ofdisease progression. Thus, based on the previous trials, it is notpossible to predict clear-cut effects in patients without bonemetastases. Therefore, ONY-P1 is significantly more effective intreating prostate cancer, for example, than the combinations of celllines reported previously (Eaton et al., BJU International (2002)89:19-26).

It is to be understood that while the invention has been described indetail by way of example and illustration for the purpose of clarity ofteaching, the foregoing description is not intended to limit the scopeof the invention. Other aspects, advantages, and modifications that areapparent to one of skill in the art in light of the teachings of thisinvention are within the scope of the following claims.

Each document cited herein is incorporated by reference in its entirety.Great Britain patent application No. 9827103.4, filed Dec. 10, 1998,PCT/GB99/04135, filed Dec. 9, 1999 and U.S. patent application Ser. No.09/857,690, filed Jun. 8, 2001 are incorporated in their entireties byreference.

1. A method of treating a prostate cancer comprising administering anallogeneic immunotherapeutic composition to a patient in need, whereinthe prostate cancer has metastasized to a tissue selected from the groupconsisting of bone, lymph node, brain and liver, and wherein theallogeneic immunotherapeutic composition comprises: a) a non-specificimmune stimulant; b) cells from an allogeneic cell line derived from aprimary non-metastasized prostate cancer biopsy; c) cells from anallogeneic cell line derived from a metastasized prostate cancer biopsy;and d) cells from an allogeneic normal prostate cell line.
 2. The methodof claim 1, wherein the non-specific immune stimulant comprises one ormore of bacille Calmette-Guerin, a Mycobacterium, Mycobacterium vaccae,Tetanus toxoid, Diphtheria toxoid, Bordetella Pertussis, interleukin 2,interleukin 12, interleukin 4, interleukin 7, Complete Freund'sAdjuvant, and Incomplete Freund's Adjuvant.
 3. The method of claim 1,wherein the non-specific immune stimulant comprises inactivatedMycobacterium vaccae bacilli.
 4. The method of claim 1, wherein thenon-specific immune stimulant comprises inactivated bacilliCalmette-Guerin.
 5. The method of claim 1, wherein the allogeneicimmunotherapeutic composition further comprises cells from at least oneother cell line derived from a prostate cancer that has metastasized toone or more tissues selected from the group consisting of the lymphnodes, bone, brain and liver.
 6. The method of claim 1, wherein theallogeneic cells are lethally irradiated to ensure that the cells arereplication incompetent.
 7. The method of claim 1, wherein theallogeneic immunotherapeutic composition further comprises acryoprotectant.
 8. The method of claim 7, wherein the cryoprotectantcomprises at least one of 10-30% v/v aqueous glycerol, 5-20% v/vdimethyl sulphoxide and 5-20% w/v human serum albumin.
 9. The method ofclaim 1, wherein the cells of b) are P4E6 cells, and the cells of c) arederived from LnCAP cell line.
 10. A method of treating prostate cancercomprising: I) providing an allogeneic immunotherapeutic compositioncomprising a) a non-specific immune stimulant; b) cells from anallogeneic cell line derived from a primary non-metastasized prostatecancer biopsy; c) cells from an allogeneic cell line derived from ametastasized prostate cancer biopsy; and d) cells from an allogeneicnormal prostate cell line; and II) administering an effective amount ofthe allogeneic immunotherapeutic composition to a patient in need,wherein the prostate cancer has metastasized to a tissue selected fromthe group consisting of bone, lymph node, brain and liver.
 11. A methodof treating a hormone-resistant prostate cancer comprising administeringan allogeneic immunotherapeutic composition to a patient in need,wherein the hormone-resistant prostate cancer has metastasized to atissue selected from the group consisting of bone, lymph node, brain andliver, and wherein the allogeneic immunotherapeutic compositioncomprises: a) a non-specific immune stimulant; b) cells from anallogeneic cell line derived from a primary non-metastasized prostatecancer biopsy; c) cells from an allogeneic cell line derived from ametastasized prostate cancer biopsy; and d) cells from an allogeneicnormal prostate cell line.